Studies on soluble fibrin in plasma IV: Isolation and characterization of the clottable proteins obtained from thrombin incubated plasma upon gelation with ethanol

Abstract

Abstract Fresh citrated or EDTA plasma, saturated with soluble fibrin by incubating with traces of thrombin gelled upon the addition of ethanol at 20°C (‘the ethanol gelation test’). Upon ultracentrifuging a thin fibrinogen-fibrin film was obtained, which readily dissolved in 0.001 N NaOH, and in 1.5 M urea pH 8, indicating mainly non-covalent bonding. Clottability was about 95%. Regular polyacrylamide electrophoresis (4% gels) showed a major component with mobility equal to fibrinogen, and two slower moving components. Immunoprecipitation in the polyacrylamide gel demonstrated FR-antigens in all three components. SDS-gel electrophoresis (3.2% gels) in urea (5 M) disclosed the slower moving components of EDTA plasma to disappear, whereas those of citrated plasma persisted. Fibrinogen-fibrin films from a patient with an inhibitor (IgG) to FSF were similar to EDTA plasma. SDS-gel electrophoresis after reduction demonstrated that films from thrombin incubated citrated plasma, in addition to the A alfa, B beta and y-chains, contained y-y dimers, whereas this was not found in EDTA or FSF-inhibitor containing plasma. Molecular weight estimates (3.2% SDS), indicated that the first fibrinogen derived component had a molecular weight of 700.000, the second 1.000.000, the third 1.300.000, indicating dimers, trimers etc. N-terminal analysis of the films demonstrated a molar content of 2 tyrosine, 0.6 glycine and 1.1 alanine. Thus, FSF-stabilized, soluble, dimeric trimeric fibrin-(ogen)-fibrin complexes may exist in citrated plasma when the ethanol gelation test is positive.